Polymeric electrophoresis media

ABSTRACT

Preformed, water-soluble, acrylamide based copolymers comprising a minor proportion of a comonomer, which comonomer contains a site for a crosslinking reaction with a selected crosslinking agent by a reaction that does not involve a free-radical vinyl addition mechanism, which copolymers have M w  and M n  values within selected ranges, can be conveniently and safely used to prepare electrophoresis gel media in situ.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a continuation of copending application Ser. No. 07/431,048filed on Nov. 2, 1989 now abandoned, which is a continuation-in-part ofU.S. application Ser. No. 339,468, filed Apr. 18, 1989, now abandoned.It is also related to the subject matter of application Ser. Nos.188,821, filed May 2, 1988 now U.S. Pat. No. 4,948,480; 339,350, filedApr. 18, 1989 now abandoned; 339,456, filed Apr. 18, 1989 now abandoned;339,469, filed Apr. 18, 1989 now abandoned and continuations orcontinuations-in-part of these applications being filed concurrentlyherewith.

FIELD OF THE INVENTION

This invention relates to a medium or element for electrophoresis. Moreparticularly, it relates to improved polymeric gel media suitable forelectrophoretic separation of biopolymers such as proteins andpolynucleic acids (DNA, RNA and their derivatives or fragments).

DESCRIPTION RELATIVE TO THE PRIOR ART

U.S. Pat. No. 4,704,198, which issued on Nov. 3, 1987, contains acomprehensive description of various aspects of electrophoresis. Asdescribed therein, and in numerous other publications, electrophoresisis based on the principle that charged molecules or substances willmigrate when placed in an electric field. Since proteins and otherbiopolymers (e.g., DNA, RNA, enzymes and some carbohydrates) arecharged, they migrate at pH values different from their isoelectricpoints. The rate of migration depends, among other things, upon thecharge density of the protein or biopolymer and the restrictiveproperties of the electrophoretic matrix or medium. The higher the ratioof charge to mass, the faster an ion will migrate. The more restrictivethe medium, the more slowly an ion will migrate. Electrophoresis has thefurther advantage of generally requiring only very small (i.e.,microgram or less) quantities of material for analysis.

Electrophoresis is generally performed in an aqueous solution or gelacross which a voltage is applied. It is the voltage gradient thatcauses the migration of the species being separated. Gradients typicallyrange from 10 volts/cm to many times higher, the magnitude depending onthe nature of the separation being formed.

In theory, separation of different proteins could be achieved readily infree solution provided that the molecules differed sufficiently in theircharge densities. However, in practice, separations in free solution aredifficult to achieve because convective disturbances produced by oroccurring during electrophoresis cause distortions of the protein bands.Resolution of the individual proteins is compromised because the bandsare broadened. Also, band broadening continues even after theelectrophoresis has been stopped because of diffusion of dissolvedsolute. Therefore, electrophoresis in free solution is rarely performed.In practice, various supporting media are used to minimize convectionand diffusion, and to effect separation both on the basis of molecularsize and of net charge.

Many support media for electrophoresis are in current use. The mostpopular are sheets of paper or cellulose acetate, agarose, starch, andpolyacrylamide. Paper, cellulose acetate, and similar porous materialsare relatively inert and serve mainly for support and to minimizeconvection. Separation of proteins using these materials is basedlargely upon the charge density of the proteins at the pH selected.

On the other hand, starch, agarose and polyacrylamide gels not onlyminimize convection and diffusion but also actively participate in theseparation process. These materials provide a restrictive medium inwhich the average size of the polymeric network opening (or average poresize) can be controlled to achieve a molecular fractionation in adesired molecular size range. In this way, molecular sieving occurs andprovides separation on the basis of both charge density and molecularsize.

The extent of molecular sieving is thought to depend on how much the gelnetwork opening size (i.e., average pore size) is larger than the sizeof the migrating particles. The average pore size of agarose gels is solarge that molecular sieving of most protein molecules is minimal andseparation of proteins in that medium is based mainly on charge density.In contrast, polyacrylamide gels can have openings whose sizes moreclosely approximate the sizes of protein molecules and so contribute tothe molecular sieving effect. Polyacrylamide has the further advantageof being a synthetic polymer which can be prepared in highly purifiedform.

With agarose, a polysaccharide, the gel is formed by casting a heated(T>50° C.) agarose solution and allowing the solution to cool. Thisprocess of gelation on cooling is similar overall, and even on amolecular basis, to the formation of gelatin gels from cooled solutionsof gelatin in water. Agarose is rarely used at concentrations higherthan 5% because such solutions are very viscous and not easily poured.Agarose is therefore widely used at concentrations <5% (w/v) for theelectrophoresis of large molecules, e.g., high molecular weightproteins, and polynucleotides.

The ability to produce gels having a wide range of polymerconcentrations (and, therefore, since the gel network opening decreaseswith increasing polymer concentration, a wide range of controlledaverage pore sizes) as well as to form pore size gradients within thegels by virtue of polymer concentration gradients, are additionaladvantages of polyacrylamide as an electrophoresis gel medium. Controlover pore size enables mixtures to be sieved on the basis of molecularsize and enables molecular weight determinations to be performed. Thesedeterminations are especially accurate if the proteins are treated witha detergent, such as sodium dodecyl sulfate (SDS), which neutralizes theeffects of inherent molecular charge so that all SDS treated molecules,regardless of size, have approximately the same charge density values.This technique is referred to as SDS-PAGE (Sodium DodecylSulfate-PolyAcrylamide Gel Electrophoresis).

The popularity of polyacrylamide-based electrophoresis gels stems notonly from the comparatively wide latitude in polymer content and buffercomposition attainable with them, but also from the high degree ofinertness in the gel with respect to both the voltages applied and thesolutes being separated, the ease with which proteins are detected onceseparated and good reproducibility with carefully prepared gels.

Conventionally, polyacrylamide gel media for use in SDS-PAGEelectrophoresis have been prepared in situ by free radical inducedpolymerization of a monomer such as acrylamide and a crosslinking agent,most commonly N,N'-methylenebisacrylamide, under oxygen-free conditionsin the presence of water, a buffer, a polymerization initiator, and apolymerization catalyst. More particularly, since such polymerizationcan be inhibited by the presence of oxygen, polyacrylamide gel media forelectrophoresis typically are prepared by a process involving:introducing a previously deoxygenated aqueous solution containingacrylamide, a crosslinking (bis) monomer, a buffer, a free radicalpolymerization initiator and a polymerization catalyst into a cellformed between two glass plates with a selected clearance (e.g., 0.15-3mm); and sealing the gel-forming solution from oxygen; whereupon thefree radical polymerization proceeds so as to prepare the desired gel.Often this is done in situ by the scientist who is to conduct theelectrophoresis.

The usual practice is to perform a free radical polymerization withacrylamide and a suitable bis monomer such asN,N'-methylenebisacrylamide (often simply referred to as "bis") in orderto obtain a gel. Such gel formation is successfully done only as severalprecautions are taken, namely: (a) very high purity starting materialsshould be used; (b) the solution of monomers and buffer should bedegassed to remove oxygen; (c) a free radical initiator and a catalystmust be quickly mixed into the degassed solution; (d) the solutionshould be quickly poured between two glass plates or down a glass tube,the lower end of which in either case is sealed to prevent leakage; and(e) the gelation should proceed with (i) oxygen largely excluded and(ii) adequate means for heat dissipation being present so that excessheat does not cause gel nonuniformities.

The cell employed for the preparation of the gel generally has a lengthof approximately 6 to 60 cm. Accordingly, the introduction of thegel-forming solution into such a long cell requires careful operation toprevent the solution from gelling before it is completely poured (whichwould prevent the preparation of a uniform polyacrylamide gel medium ofthe desired length). Thus, the preparation of a polyacrylamide gelmedium for electrophoresis having the desired dimensions and consistencyrequires a great deal of skill and care, as well as keeping the solutionfree from oxygen.

Precautions are also required in handling the monomers since bothacrylamide and bis have been identified as known neurotoxins andsuspected carcinogens.

There are several alternatives to the above-described procedure wherebythe user makes electrophoresis gels by free radical polymerization andcrosslinking in situ. These include (a) the use of preformed gels incassettes and (b) the use of preformed gels on flexible supports. Witheither of these alternatives, however, some operating freedom orflexibility with regard to gel size, polymer content in the gel andbuffer content is lost. Also--especially with precast gels in cassettesmade by free radical polymerization and crosslinking--there generallyremain, after completion of the gel formation reaction, some unreactedmonomers, initiator by-products and catalyst. The presence of suchspecies poses some toxicological hazards to the user and may interferewith the electrophoretic separation to be performed. Also, such precastgels have been found to have limited shelf lives.

U.S. Pat. No. 4,582,868, among others, describes the crosslinking ofacrylamide-rich copolymers to form electrophoresis gel media by anon-free radical induced mechanism that does not require exclusion ofoxygen. Typically, copolymers of acrylamide and a monomer that affordssites for subsequent non-free radical initiated crosslinking bytreatment with a crosslinking agent, for example, an acrylamidederivative such asN-[3-(2-chloroethylsulfonyl)propionamidomethyl]acrylamide, ##STR1## anacrylate derivative such as2-[3-(2-chloroethylsulfonyl)propionyloxy]ethyl acrylate, ##STR2## anactive ester such asN-[2-(ethoxycarbonylmethoxycarbonyl)ethyl]acrylamide:

    CH.sub.2 =CHCONHCH.sub.2 CH.sub.2 COOCH.sub.2 COOC.sub.2 H.sub.5

are prepared, in accordance with U.S. Pat. No. 4,582,868, by a freeradical initiated polymerization in the absence of oxygen. Thereafter,in a separate procedure, which can safely be performed in the presenceof oxygen, the chloroethylsulfonyl or other pendent reactivegroup-containing polymers are crosslinked by reaction at a suitable pHwith a bis-nucleophile crosslinking agent such as a diamine or adithiol. In this regard, it is noted that electrophoresis often isperformed at pH values that facilitate dehydrohalogenation of thechloroethylsulfonyl groups. If the vinylsulfonyl groups so formed arenot all reacted with the intended crosslinking agent, they could reactwith amino groups on dissolved proteins during electrophoresis. Suchreaction would artifactually retard the electrophoretic migration ofproteins and consequently give misleading electrophoresis resultsvis-a-vis the results obtained with electrophoresis gels formed by thefree radical polymerization of acrylamide and bis alone. Therefore,enough crosslinking agent should be used to assure complete reaction ofthese groups.

Despite the availability of the above-described alternatives,electrophoresis media are still generally prepared by the polymerizationof vinyl monomers at the time of use. This necessarily involves exposureof the operator to monomers prior to use and to residual monomers duringuse. Such monomers are suspected carcinogens, and at least some areknown to be neurotoxins.

Although bis is the most widely used crosslinker for acrylamide-basedelectrophoresis gels, bis-crosslinked gels generally cannot beresolubilized. The ability to solubilize the gel after performingelectrophoresis is advantageous in that it enables one to recover aresolved species from the gel. The use of an alternative, cleavablecrosslinker such as diallyltartardiamide (DATD) ##STR3## permits theresearcher to achieve such recovery. After electrophoresis, each portionof the gel to be solubilized is excised and treated in a dilute solutionof periodic acid. The --CHOH--CHOH-- linkage in the middle of thecrosslink is cleaved in the periodic acid solution and a solution of thethus solubilized polymer is produced. From this solution one can easilyrecover the resolved species for further experiments. Obviously, itwould be advantageous to provide researchers with the option of gelsolubilization after electrophoresis.

Also, if electrophoresis is to be performed soon after gelation, thechemical reaction (i.e., crosslinking) responsible for gelation must becompatible with the buffers present for electrophoresis. Gelelectrophoresis is often performed at pH values ranging from 5 to 9.

A particularly popular system for the determination of molecular weightsof proteins by electrophoresis was described by Laemmli, Nature, 227:680(1970). In this system, two gels are used, one directly above the other,with a multi-phasic buffer system. The upper (or stacking) gel is at pH6.8, which is achieved with tris(hydroxymethyl)aminomethane to which HClhas been added to lower the pH. The stacking gel has a low polymerconcentration (generally from 4 to 6% w/v). Its purposes are (a) toprovide a medium onto which samples can be loaded in discrete lanes and(b) to concentrate all species in a particular sample at the interfacebetween the stacking gel and the lower (or resolving) gel. In meetingobjective (b), the solutes, which are generally sodium dodecylsulfate(SDS)-denatured proteins, are "stacked" together (or very nearly so)i.e., are concentrated at the interface of the two gels, just beforeentering the resolving gel. For the stacking to occur effectively, theproteins, rather than the buffer, should carry most of the current andthere should be no molecular size separation.

Molecular size separation occurs in the resolving gel, where the buffercarries most of the current and the solutes migrate at a velocitydetermined by the voltage gradient and the retardation due to the poresize distribution of the crosslinked polymer gel. The pH in theresolving gel is typically 8.8 and the polymer concentration is usuallyat least 10% (w/v).

In summary, the popular Laemmli procedure requires two gels, the lowerof which (resolving gel) is larger (thereby providing a longer path forsolutes to tranverse) and contains a higher concentration of the gelledpolymer, and therefore a smaller average pore size than that of theupper, or stacking gel. The conditions recommended by Laemmli are:

    ______________________________________                                                 Stacking Gel                                                                             Resolving Gel                                             ______________________________________                                        pH         6.8          8.8                                                   buffer     0.125M Tris.HCl                                                                            0.375M Tris.HCl                                       gel conc., 4-6          >10                                                   % (w/v)                                                                       ______________________________________                                    

Despite the various advances in electrophoresis technology, there isstill a need for further improvements so that one not only can coat thesolutions to be gelled in the presence of oxygen but also can easily andsafely (a) pour such solutions into tubes or between plates, (b) formgels quickly and (c) solubilize gels or portions thereof for examplerecovery after electrophoresis.

SUMMARY OF THE INVENTION

The present invention provides more convenient, safer means than wereheretofore available for preparing an acrylamide-based electrophoresisgel that permits the operator to overcome several disadvantages found inthe prior art. In accordance with the present invention, we havediscovered that the weight average molecular weight M_(w) and the numberaverage molecular weight M_(n) are among the critical parameters thatmust be properly selected in order for a preformed polymer that issubsequently crosslinked with a chemical crosslinking agent that doesnot involve a free-radical vinyl addition mechanism, first to produce agellable polymer solution for forming a resolving electrophoresis gelthat (1) is easily poured into a slot (mold, tube, or between glassplates) no greater than 0.15 cm thick, (2) has a short gel time (i.e.,about 4 to 10 minutes; by "gel time" is meant the time from addition ofthe crosslinking agent until the solution cannot be poured easily), but(3) remains pourable into said slot for a sufficient time to fill anelectrophoresis mold of 14×14×0.15 cm (which takes about 4 to 6minutes); and second, within about 10 minutes to 2 hours after additionof the crosslinking agent, to produce a firm gel having (1) sufficientcrosslink density to afford good molecular sieving duringelectrophoresis, (2) good resolution during electrophoresis, and (3)sufficient physical integrity to permit manual removal of the hardenedgel from the mold shortly after electrophoresis as well as its handlingwithout causing detrimental flow, compression, fracture, strech, tear,or disintegration of the gel.

The requirements of M_(w) and M_(n) in said preformed polymer componentof the electrophoresis media are:

1) M_(w) is small enough that the addition of 1.25 to 1.5 times thestoichiometric amount of the selected crosslinking agent will not raisethe viscosity of the mixture so much that the media cannot be pouredinto a 0.15×14×14 cm mold within about 4 to 10, preferably about 6 to 8minutes after addition of the crosslinking agent,

2) M_(n) is enough to provide a crosslink density sufficient to form agel after about 10 minutes after addition of 1.25 to 1.5 times thestoichiometric amount of the selected crosslinking agent, said gelultimately having sufficient integrity within about 2 hours afterpouring to be removed from the mold and handled gently without tearingor falling apart, and

3) the number of equivalents of crosslinking sites per gram of polymeris in the range of 0.45(10⁻⁴) to 14(10⁻⁴), preferably at least 2/M_(n) ;more preferably from about 2.25(10⁻⁴) to 10(10⁻⁴), most preferably about4(10⁻⁴) to 7(10⁻⁴).

More particularly, a copolymer of the present invention for preparing aresolving gel for electrophoresis is a water soluble vinyl additioncopolymer derived from a mixture of monomers comprising from 85-98 molepercent, preferably 90 to 97 mole % of a monomer selected fromacrylamide and the (N-substituted acrylamides wherein the N-substituentis an alkyl group having from 1 to 5 carbon atoms, from 2 to 15 molepercent, preferably 3 to 10 mole percent, of a vinyl monomer having areactive group selected from the group consisting of a) active halogengroups; b) activated 2-substituted ethylsulfonyl or activatedvinylsulfonyl groups; c) epoxy groups; d) isocyanate groups; e)aziridine groups; f) aldehyde groups; g) 2-substituted ethylcarbonylgroups; and h) succinimidoxycarbonyl groups; and from 0 to 12 molepercent of one or more other polymerizable nonionic vinyl monomersselected from styrene monomers, acrylic monomers, methacrylamidemonomers and N-substituted acrylamide monomers wherein the substituentcontains at least 6 carbon atoms, said water soluble copolymer having anumber average molecular weight, M_(n), of at least about 7,000,preferably from about 7,000 to about 30,000, and a weight averagemolecular weight, M_(w), of less than about 100,000, preferably fromabout 25,000 to about 100,000.

In another aspect, this invention also relates to a new stacking gelcomposition comprising polymers of the same chemical composition asdescribed for the resolving gel but having a much greater value forM_(w). M_(n) is also generally higher but this parameter is not ascritical as M_(w) in the copolymer for the stacking gel. These molecularweights are each significantly higher than those of the resolving gelwhen polymers of exactly the same chemical composition are used. TheM_(n) is preferably greater than about 50,000 and M_(w) is greater thanabout 100,000, preferably greater than about 150,000. The gel time issomewhat longer than that of the resolving gel because stacking gels areformed at a lower concentration (4-6% vs about 12%) and a lower pH(6.0-8.0) than resolving gels.

In yet another embodiment, the copolymer of the present invention,whether in the form of a resolving gel, stacking gel or both, isprovided in a kit for preparing an electrophoresis gel, this kitcomprising the copolymer and, in a separate container from thecopolymer, a suitable crosslinking agent for crosslinking the copolymerby a reaction that does not involve a free-radical initiated vinyladdition mechanism. Optionally, the kit may also contain a selectedbuffer and other suitable ingredients for incorporation into theelectrophoresis medium.

DETAILED DESCRIPTION OF THE INVENTION

A preferred preformed crosslinkable polymer useful for preparing aresolving gel for electrophoresis in accordance with this invention ispoly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] (weightratio of acrylamide to comonomer: 90/10; mole ratio: 96.5/3.5) used at aconcentration of about 12% (w/v) in the gel. It has a preferred M_(w) ofabout 4.6×10⁴ ; a preferred M_(n) of about 1.7×10⁴ ; and preferablyabout 4.59×10⁻⁴ equivalents of crosslinking site per gram.

In addition to the above preferred polymer, any water-soluble vinyladdition acrylamide copolymer having the requisite M_(w), M_(n), andequivalents of crosslinking site per gram of polymer would be useful inthe practice of this invention, especially copolymers of the structure:

    --A).sub.x --B).sub.y --D).sub.z                           (I)

wherein

--A-- represents recurring units derived from acrylamide andN-substituted acrylamide wherein said substituent has less than about 6carbon atoms;

--B-- represents recurring units derived from vinyl monomers containingcrosslinkable sites selected from:

a) active halogen groups;

b) activated 2-substituted ethylsulfonyl or activated vinylsulfonylgroups;

c) epoxy groups;

d) isocyanate groups;

e) aziridine groups;

f) aldehyde groups;

g)2-substituted ethylcarbonyl groups; and

h) succinimidoxycarbonyl groups;

--D-- represents recurring units derived from any other nonionicmonomers including styrene monomers, acrylic monomers, methacrylamidemonomers, and substituted acrylamide monomers wherein said substituentshave 6 or more carbon atoms; and x, y, and z represent mole percents, xbeing 85 to 98, preferably 90 to 97 mole %; y being 2 to 15, preferably3 to 10 mole % and z being 0 to 12, preferably 0 to about 3 mole %.

Examples of suitable acrylamide monomers (A) for inclusion in thecopolymers of the present invention include acrylamide,N-isopropylacrylamide, N-hydroxymethylacrylamide,N-(1,1-dimethyl-3-oxobutyl)acrylamide, N-methylmethacrylamide,2-acrylamido-2-hydroxmethyl-1,3-propanediol, methacrylamide,3-(3-dimethylaminopropyl)acrylamide, N,N-dimethylacrylamide,N,N-diethylacrylamide, N-isopropylmethacrylamide, and3-(2-dimethylaminoethyl)acrylamide. Particularly preferred is(unsubstituted) acrylamide.

The --B-- recurring units can contain any of the reactive groups a) toh) to provide the required concentration of sites for crosslinking forpreparing electrophoresis gel media in accordance with the invention.All of these groups react readily with difunctional or polyfunctionalamino and/or sulfhydryl group-containing crosslinking agents.

While free or ionized pendent carboxyl groups could afford crosslinkingsites on the polymer, they should be avoided because, being ionic, anyunreacted carboxyl groups (or other ionic groups) could interfere withelectrophoretic separations in SDS-PAGE electrophoresis.

One preferred class of monomers which provide the requisite reactivegroups is the monomers containing an active halogen atom which readilyreacts with amine and sulfhydryl groups. Examples of monomers having anactive halogen atom include vinyl chloracetate, vinyl bromoacetate,haloalkylated vinyl aromatics (for example, chloromethylstyrene andbromomethylstyrene), haloalkyl acrylic and methacrylic esters (forexample, chloroethyl methacrylate, 3-chloro-2-hydroxypropyl methacrylateand 3-chloropropyl acrylate), N-(3-chloroacetamidopropyl)methacrylamide,2-chloroacetamidoethyl methacrylate, 4-chloroacetamidostyrene, 3- and4-chloroacetamidomethylstyrene,N-[3-(N'-chloracetylureido)propyl]methacrylamide,2-(N'-chloroacetylureido)ethyl methacrylate,4-(N'-chloroacetylureido)styrene,4-(N'-chloroacetylureidomethyl)styrene, and others known to thoseskilled in the art.

Another useful class of monomers comprises those having activated2-substituted ethylsulfonyl and vinylsulfonyl groups. A number ofrepresentative monomers having the latter groups are known in the art,including those disclosed in U.S. Pat. Nos. 4,161,407 and 4,548,870.

Preferred activated 2-substituted ethylsulfonyl and vinylsulfonylmonomers can be represented by the formula (II): ##STR4## wherein R ishydrogen or substituted or unsubstituted alkyl (generally of 1 to 6carbon atoms, such as methyl, ethyl, isopropyl or hexyl). Preferably, Ris hydrogen or methyl.

R¹ is --CH═CHR² or --CH₂ CH₂ X wherein X is a leaving group which isdisplaced by a nucleophile or is eliminated in the form of HX bytreatment with a base (such as halo, acetoxy, alkylsulfonyloxy such asmethylsulfonyloxy, arylsulfonyloxy such as p-tolylsulfonyloxy, andtrialkylammonio, for example, a trimethylammonio salt, or a pyridiniosalt). R² is hydrogen, substituted or unsubstituted alkyl (generally of1 to 6 carbon atoms as defined for R), or substituted or unsubstitutedaryl (generally of 6 to 12 ring carbon atoms, such as phenyl, naphthyl,xylyl or tolyl). Preferably, R¹ is --CH₂ CH₂ X. This group, which is anactivated 2-substituted ethyl group, can be substituted with any groupwhich does not impair the displacement of the leaving group X.

L is a linking group which can be a substituted or unsubstitutedalkylene generally having 1 to 20 carbon and hetero atoms in thebackbone. This definition of alkylene is meant to include alkylenegroups interrupted or terminated with oxy, thio, --NR³ -- [wherein R³ ishydrogen, substituted or unsubstituted alkyl of 1 to 6 carbon atoms(such as methyl, chloromethyl or 2-hydroxyethyl) or substituted orunsubstituted aryl of 6 to 12 carbon atoms (such as phenyl, naphthyl,xylyl, or tolyl)], ester (--COO--), amide (--CONH--), urylene ##STR5##sulfonyl (--SO₂ --), carbonate, sulfonamide, azo, phosphono or othersimilar groups. Representative alkylene groups include methylene,ethylene, isobutylene, hexamethylene, carbonyloxyethoxycarbonyl,methylenebis(iminocarbonyl), carbonyloxydodecylenecarbonyloxyethylene,carbonyliminomethyleneiminocarbonyliminoethylene,carbonyliminomethyleneiminocarbonylethylene and other groups describedor suggested by U.S. Pat. Nos. 4,161,407 and 4,548,870, noted above.

L can also be substituted or unsubstituted arylene generally having 6 to12 ring carbon atoms. Representative arylene groups include phenylene,tolylene, naphthylene and others noted in the patents mentioned above.Also included in this definition of L are divalent groups which arecombinations of one or more of each of the alkylene and arylene groupsdefined above (for example, arylenealkylene, alkylenearylenealkylene andothers readily determined by one of ordinary skill in the art).Preferably L is unsubstituted phenylenealkylene, phenylenealkylenesubstituted with one or more alkyl groups (as defined for R), alkoxygroups (generally of 1 to 6 carbon atoms, for example, methoxy, propoxyor butoxy) or halo groups, orcarbonyliminomethyleneiminocarbonylethylene.

Representative 2-substituted ethylsulfonyl and vinyl sulfonyl monomersfrom which --B-- can be derived include m andp-(2-chloroethylsulfonylmethyl)-styrene, m and p-[2-p-tolylsulfonyloxy)ethylsulfonylmethyl]styrene, m andp-vinylsulfonylmethylstryene,N-[p-(2-chloroethylsulfonylmethyl)phenyl]acrylamide, andN-[2-(2-chloroethylsulfonyl)ethylformamidomethyl]acrylamide.

Other monomers which can be incorporated in the polymers to provide therequisite reactive groups include monomers containing epoxy groups (suchas glycidyl acrylate, glycidyl methacrylate, vinyl glycidyl ether ormethallyl glycidyl ether), monomers containing isocyanate groups (suchas isocyanatoethylacrylate, isocyanatoethyl methacrylate, orα,α-dimethylmetaisopropenylbenzyl isocyanate), monomers containing anaziridine group [such as vinylcarbamoylaziridine, acryloylaziridine,methacryloylaziridine, and 2-(1-aziridinyl)ethyl acrylate], monomerscontaining aldehyde groups (such as vinyl benzaldehyde or acrolein), or2-substituted ethylcarbonyl containing monomers (such as 2-chloroethylacrylate, 2-chloro-ethyl methacrylate, 2- methylsulfonyloxyethylmethacrylate, and 2-p-tolylsulfonyloxyethyl acrylate).

The foregoing polymers can be crosslinked with agents having two or moreamino, mercapto, sulfinic acid, or phenolic hydroxy groups such asethylenediamine, 1,3-propanediamine, 1,3-propanedithiol, dithiothreitol,dithioerythritol, 1,5-pentanediamine, hexamethylenediamine,diethylenetriamine, triethylenetetramine, propylenediamine,di(aminomethyl)ether, 1,8-diamino-4-(aminomethyl)octane,xylylenediamine, hydroquinone, bisphenol A, bisphenol sulfone,1,4-butanedisulfinic acid, benzenedisulfinic acid, thioethanolamine,p-aminothiophenol, and butylenediamine.

Our preferred reactive group for --B-- is the chloroacetyl, especiallyin the form of a chloroacetamido group. This group is electrophilic andwould be expected to react with a nucleophile such as an amine or athiol. We have found that the reaction with a thiol occurs much morerapidly than with an amine at the pH values (7-10) of most interest forelectrophoresis. Thus we have found in one preferred embodiment thatdithiothreitol (DTT), HSCH₂ (CHOH)₂ CH₂ SH, is a very effectivecrosslinking agent for this class of polymers. The advantages of DTT,besides the rapid crosslinking reaction even in the presence of aprimary amine (Tris buffer), include (a) water solubility, (b) lowtoxicity, and (c) susceptibility to post-electrophoresis reaction withperiodic acid to solubilize the gel or selected portions thereof.

The chloroacetamido-thiol reaction is pH sensitive and produces HCL as aby-product of the reaction: ##STR6##

Consequently, we have found it advantageous to adjust to 7.8 and 9.4,respectively, the pH's of the Tris·HCl solutions used in the stackinggel and resolving gel buffers. These adjustments increase the rate ofreaction and compensate for the generation of HCl. We have found thatthe electrophoretic separation of SDS-complexed proteins on DTTcrosslinkedpoly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] copolymerscompares very favorably to the separation obtained on acrylamide/bisgels.

Selection and incorporation of suitable buffer is well within theknowledge of skilled workers in the electrophoresis art and depends uponthe materials to be separated by the electrophoresis process in whichthe medium is to be employed. Such buffers and bases for selecting themare described, for example, in Andreas Chrambach, "The Practice ofQuantitative Gel Electrophoresis," VCH Publishers, Deerfield Beach,Fla., U.S.A. (1985), and U.K. Laemmli, Nature, 227:680, (1970).

The preferred units --B-- bearing the chloracetamido group (as is truefor any recurring unit --B-- in the polymers of the present invention)should be present only to the extent needed to provide the desireddegree of crosslinking density in the polymeric gel. Less than thenecessary amount would lead to gels in which the crosslink density istoo low, whereas greater amounts than needed would detract from the theacrylamide-like character that we seek. We have found that the crosslinkdensity and molecular weight requirements are satisfied if the copolymercontains on a weight basis 10% N-(3-chloroacetamidopropyl)methacrylamidemonomer (on a mole basis this is 3.5%N-(3-chloroacetamidopropyl)methacrylamide).

With 10% (by weight) N-(3-chloroacetamidopropyl)methacrylamide in theresolving gel (G_(R)) copolymer, the number of equivalents ofcrosslinking site per gram (y) can be computed as ##EQU1##

Knowing y and the molecular weight between crosslinks (M_(c)) desiredpermits the estimation of the minimum value of M_(n) for the startingcopolymer consistent with achieving the crosslink density sought.##EQU2##

The preferred weight average molecular weight M_(w) is not estimated sodirectly. We have found, nevertheless, that the elapsed time necessaryto form a gel after addition of crosslinking agent to an aqueoussolution of polymer in accordance with the invention decreases withincreasing M_(w) (as expected) and that for a 12% (w/v) polymerconcentration in the resolving gel one can achieve a gelation time ofsix minutes (time between crosslinker addition and gelation) if M_(w) isapproximately 4.6(10⁴).

In a preferred embodiment, the new resolving gel (G_(R)) described isused in combination with a polymeric stacking gel (G_(S)). Forcompatibility, the monomers from which the stacking gel copolymer ismade should be the same as, or similar to, those used for the resolvinggel. Preferably, acrylamide comprises at least 90 mole percent of themonomer mix. Consequently, the molecular weights, M_(n) and M_(w) of thestacking gel copolymers must be significantly greater than those of thecorresponding resolving gel copolymers; preferably, their M_(w) isgreater than about 100,000, more preferably greater than about 150,000,suitably about 200,000 or 300,000 and their M_(n) is greater than about50,000.

The rate of gelation can be expected to increase as (a) theconcentration of copolymer in the gel, (b) the molecular weight of thestarting copolymer (particularly M_(w)), (c) the number of crosslinkingsites present per gram of copolymer, and (d) the pH of the solution,increase.

To prevent molecular sieving by the stacking gel, the stacking gelpolymer is present at a lower concentration than that of the resolvinggel polymer since porosity of the gel is a function of polymerconcentration (i.e., average pore size decreases with increasing polymerconcentration).

Typically, concentration of the preformed polymer will be from about 8to about 14%, preferably 10 to 12% (w/v) for the resolving gel and fromabout 1.5 to about 6%, preferably 2 to 5%, more preferably 2.5 to 4%(w/v) for the stacking gel. For example, the preferred polymer,poly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] (weightratio of acrylamide to comonomer: 90/10; mole ratio: 96.5/3.5), isemployed at a concentration of about 12% to make a resolving gel, andabout 4% to make a stacking gel. However, to ensure gel formation, themolecular weights M_(n) and M_(w) are both higher in the stacking gelpolymer than in the resolving gel polymer.

The preferred polymer,poly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] weightratio of acrylamide to comonomer: 90/10; mole ratio: 96.5/3.5),preferably has the following molecular weights when used for bothpurposes:

    ______________________________________                                                      -- M.sub.n                                                                            -- M.sub.w                                              ______________________________________                                        Resolving Gel   >0.7(10.sup.4)                                                                          <10.0(10.sup.4)                                     (G.sub.R)                                                                     Stacking Gel      >5(10.sup.4)                                                                          >10.0(10.sup.4)                                     (G.sub.s)                                                                     ______________________________________                                    

As indicated earlier, these preferred molecular weights should besimilar for any other polymers of structure I that are crosslinkedsimilarly. This is because: 1) the bulk of the polymer composition isderived from acrylamide or modestly substituted acrylamides, 2) thefraction of crosslinking sites per molecule should be similar, and 3)the molecular weight of polymer backbone between crosslinks should alsobe similar. It is expected therefore that the useful polymers of theinvention for forming resolving gels should have an M_(n) of about 7,000to 30,000 and an M_(w) of about 25,000 to 100,000; and those for formingstacking gels should have an M_(n) of about 50,000 to 300,000 and anM_(w) of about 100,000 to 1,000,000.

Regarding the number of crosslinking sites present on the polymer,whether used as ther resolving or stacking gel, the crosslinkingreactions should ideally be done using equal equivalent (i.e.,stoichiometric) amounts of reactive crosslinking agent and sites on thepolymer. However, we have found it advantageous to use 25-50% moredithiothreitol (or other suitable crosslinking agent) on a chemicalequivalency basis than there are crosslinking sites present. Thebenefits of using 1.25 to 1.5 times the just-required stoichiometricamount of crosslinking agent are (a) better overall crosslinking asmanifested by lower degrees of gel swell in high purity water, and (b)assurance that most (if not all) of the electrophilic groups have beenreacted. (Unreacted electrophilic groups at potential crosslinking sitescould react with nucleophilic groups on proteins and thereby confoundthe electrophoretic separation process.)

The molecular weights M_(n) and M_(w) can be varied by methods known tothose skilled in the synthetic polymer chemistry art. For example, themolecular weights can be decreased by increasing the amount of initiatorused, increasing the amount of chain transfer agent used, decreasing themonomer concentration, and increasing the reaction temperature. They canalso be varied by selection of the particular chain transfer agentand/or initiator.

The number of equivalents of crosslinking sites per gram of polymer canbe varied by adjusting the concentration of monomer(s) from which the'B-- recurring units of structure I are derived. The following examplesillustrate the practice of this invention:

EXAMPLE 1

Preparation of a low molecular weight copolymer with an adequate levelof crosslinking site to form a resolving gel in about four minutes.

To a 60° C. solution of 2,2'-azobis(methylpropionitrile) (1.0 g) inMilli Q water (i.e., water purified with a Millipore C/N ZD40115-84unit) (200 mL), isopropanol (20 mL) and conc. sulfuric acid (0.5 g),which had been purged with nitrogen, was added the following solution,which also had been purged with nitrogen: electrophoresis gradeacrylamide (64.8 g; 0.91 moles),N-(3-chloroacetamidopropyl)methacrylamide (7.2 g, 0.033 moles), Milli Qwater (600 mL), and isopropanol (60 mL), the chain transfer agent. Thiswas added to the first solution dropwise and under nitrogen over a2-hour period. After the addition was complete, the resultant solutionwas held for 5 hours more at 60° C. and then allowed to stand at ambienttemperature overnight. The next day the solution was concentrated at 40°C. on a rotary evaporator to a volume of about 500 mL. The concentratedsolution was then added to 8 liters of reagent grade methanol, withstirring, to precipitate the copolymer. The precipitate was filtered andwashed in an additional 4 liters of reagent grade methanol, then driedin a vacuum oven with a nitrogen bleed at 30°-35° C. The yield was 95%based on the original recipe. The inherent viscosity of the copolymer,as determined with a 0.25% solution of the copolymer in aqueous 1.0molar NaCl at 25° C., was 0.35 dl/g. Cl analysis: theory, 1.55%; found1.46%. Molecular weight analysis by aqueous size exclusionchromatography showed M_(n) =1.51(10⁴) and M_(w) =9.41(10⁴).

A polymer solution cast from a 12% solution of this copolymer at pH 9.4began to gel (to a resolving gel) four minutes after addition of thecrosslinker (dithiothreitol--44.2 mg per gram of copolymer).

In this and the following examples, the molecular weight averages of theacrylamide/N-(3-chloroacetamidopropyl)methacrylamide copolymers wereestimated using an aqueous gel permeation chromatography system in which(a) the fractionation was accomplished with four TSK-GEL (type PW)columns of 6000, 5000, 3000 and 2000 Angstrom permeability limits (AltexScientific, 1780 Fourth St., Berkeley, Calif. 94710), (b) the eluent was0.05M Na₂ SO₄ in 5% ethylene glycol-in-water (v/v), (c) the calibratingstandards were Shodex STANDARD P-82 polysaccharides of 853, 380, 186,100, 48.0, 23.7, 12.2 and 5.8 kDa (Showa Denko K.K., 280 Park Ave., 27thFloor West Building, New York, N.Y. 10017) used at 0.1% (w/v)concentration, (d) the flow rate was 1.5 ml/min and (e) the detection ofsolute in the column effluent was done refractometrically.

EXAMPLE 2

Preparation of a high molecular weight copolymer with adequatecrosslinking site level for forming stacking gels in approximately 40minutes.

To a 50° C. solution of (NH₄)₂ S₂ O₈ (0.75 g) and NaHSO₃ (0.0375 g) inMilli Q water (100 mL) which had been purged of dissolved oxygen bybubbling with nitrogen, was added dropwise in a nitrogen atmosphere over2 hours the following solution: electrophoresis grade acrylamide (64.8g; 0.91 moles), N-(3-chloroacetamidopropyl)methacrylamide (7.2 g; 0.033moles), and NaHSO₃ (0.225 g) in Milli Q water (300 mL). The resultantsolution was held at 50° C. for an additional hour after the addition ofthe monomer-containing solution was finished. Then 250 mL more of MilliQ water were added with stirring. The copolymer was precipitated byadding the above solution to 8 liters of reagent grade methanol. Theprecipitate was filtered, washed and dried (oven at 30°-35° C.). Theyield based on the original recipe was 100% and the inherent viscosityof a 0.25% solution of the copolymer in aqueous 1.0 molar NaCl was 1.13dl/g, which indicates a very high molecular weight copolymer. Clanalysis: theory, 1.55%; found, 1.40%. When a stacking gel is cast froma 4% solution of this copolymer at a pH of 7.8 using dithiothreitol asthe crosslinker (44.2 mg per gram of copolymer), gelation occurs 40minutes after crosslinker addition.

EXAMPLE 3

Preparation of electrophoresis gel

An electrophoresis gel was made withpoly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] (weightratio of acrylamide to comonomer: 90/10; mole ratio: 96.5/3.5)copolymers disclosed herein. This gel included a lower resolving gelmade from poly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide](weight ratio of acrylamide to comonomer: 90/10; mole ratio: 96.5/3.5)of M_(n) =17.8 kDa and M_(w) =78.2 kDa and a stacking gel from apoly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] (weightratio of acrylamide to comonomer: 90/10; mole ratio 96.5/3.5) of Example2 (M_(w) ≃500 kDa). Each copolymer was crosslinked with dithiothreitolused at 125% of the stoichiometric amount based on the number ofcrosslinking sites present. The pH of, and buffers used in, the gels andthe electrode chambers are summarized in Table I.

                  TABLE I                                                         ______________________________________                                                  Buffer                                                              Location    pH      Composition                                               ______________________________________                                        Cathode     8.3     0.025M Tris, 0.192M glycine,                                                  0.1% SDS                                                  Stacking gel                                                                              7.8     0.125M Tris.HCl                                           (4% polymer)                                                                  Resolving gel                                                                             9.4     0.375M Tris.HCl                                           (12% polymer)                                                                 Anode       8.3     same as cathode except SDS was                                                omitted and 0.1M sodium                                                       acetate beneficially added                                                    (These variations from cathode                                                conditions are optional.)                                 ______________________________________                                         Tris = Tris(hydroxymethyl)aminomethane                                        SDS = sodium dodecyl sulfate                                             

Tris=Tris(hydroxymethyl)aminomethane

SDS=sodium dodecyl sulfate

In electrophoresis experiments conducted according to the Laemmliprocedure with the buffer compositions given in Table I, thedithiothreitol crosslinkedpoly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] (weightratio of acrylamide to comonomer: 90/10; mole ratio 96.5/3.5) copolymersof the present invention perform comparably to gels prepared fromacrylamide and N,N'-methylenebisacrylamide. Specifically, the gelsprepared from copolymers of this invention permit good electrophoresisseparations, with SDS-complexed proteins with molecular weights fromabout 14.4 kDa to about 200 kDa appearing at the anode and cathode endsof the gel, respectively, after electrophoresis under conditions ofvoltage and time ordinarily used by those skilled in the art ofelectrophoresis for SDS-PAGE electrophoresis on acrylamide/bis gelmedia. Not only is the degree of separation comparable to that achievedwith acrylamide/bis-based gels but the sharpness of the separated bandsis also very good. These results can be achieved with gels in theso-called "mini" format (0.15 cm×7 cm×8 cm) (thickness×height×width) andin the popular larger format (0.15 cm×16 cm×14 cm).

EXAMPLE 4

Gel solubilization after electrophoresis.

Tas et al. report the solubilization of acrylamide gels crosslinked withDATD or DHEBA with periodic acid. The solubilization of the gel is basedon the existence of a --CHOH--CHOH-- group in the crosslink chain. Theperiodic acid breaks the C--C bond and allows the gel to be solubilized[Tas et al., Analytical Biochemistry, 100:264-270 (1979)].

A portion of a gel made from dithiothreitol andpoly[acrylamide-co-N-(3-chloroacetamidopropyl)methacrylamide] (weightratio of acrylamide to comonomer: 90/10; mole ratio: 96.5/3.5) in a pH9.4 Tris-HCl buffer according to the practice of this invention wasimmersed in distilled water for 24 hours after the crosslinking reactionwas complete. Then, this gel was immersed in a 10 mM solution ofperiodic acid in water. Within one hour after immersion in the diluteperiodic acid solution the gel had vanished and become a solution of thecopolymer.

Another portion of the same gel was immersed in water and this portionwas entirely intact after such immersion for one hour. Hence, thelinkage due to dithiothreitol is cleaved by periodic acid and thepolymer that was originally gelled by the crosslinking reaction can beresolubilized by immersion in a dilute periodic acid solution.

Formation of the crosslinked gel in the presence of the Tris-HCl bufferduring the DTT crosslinking reaction is proof of the faster reaction ofthe DTT sulfhydryl groups than of the Tris-HCl amino groups. If thiswere not so, the polymer would have been capped with the monoaminerather than crosslinked to a gel with the bismercaptan.

EXAMPLE 5

a) Synthesis of high molecular weight copolymer for very low polymercontent gels.

Into a reaction vessel held at 50° C. which initially contained anitrogen-purged solution of 1.5 gram ammonium persulfate dissolved in400 ml high purity water, were pumped (i) a (previously nitrogen-purged)solution of 259.2 grams of electrophoresis grade acrylamide, 28.8 gramsof N-(3-chloroacetamidopropyl)methacrylamide, and 1200 ml of high puritywater and (ii) another (nitrogen-purged) solution of 0.525 g sodiumbisulfite and 10 ml of high purity water. Solutions (i) and (ii) wereadded to the reaction vessel over a period of 41 minutes and thecombined solutions were held for four hours at 50° C., after which thereaction mixture was permitted to cool to room temperature. Beforefurther use, an additional 500 ml of high purity water were added tothis solution, with thorough mixing, to yield a solution that contained12.5 to 13% (w/v) high molecular weight copolymer.

b) Forming a gel for DNA electrophoresis

To form a gel of very low [2.5% (w/v)] polymer concentration, 1.88 ml ofthe final solution from Example 5a, 0.5 ml of concentrated TBE buffer[0.2 moles tris(hydroxymethyl)aminomethane (Tris), 0.022 moles boricacid, 0.002 moles ethylenediaminetetraacetic acid and enough high puritywater to make 100 ml of solution], 7.59 ml high purity water, 5microliters of ethidium bromide in water solution (1 mg dye per mlsolution) and 26.5 microliters of dithiothreitol in water solution (0.5g DTT per ml) were mixed well and poured into a shallow plastic tray,the ends of which had been taped in order to contain the copolymersolution. Just after pouring, a multi-toothed plastic well former wasinserted into the gel perpendicular to the plane of the gel andperpendicular to the direction of electrophoresis. This assembly waspermitted to stand in a covered container (so as to retard evaporationof water) for three hours, after which the gel was overlaid with a TBEsolution made by a twenty-fold dilution of the concentrated TBE solutionpreviously described. Then the plastic lane former was carefully removedand the tray (from which the tape at each end was also removed) wasinserted into a horizontal electrophoresis cell, to which diluted TBEbuffer was added so that the face of the gel was about one mm under thesurface of the buffer. Then solutions of undenatured DNA fragments(double-stranded DNA) were loaded into the wells. Electrophoresis at 100V (voltage gradient about 10 V/cm) for 73 minutes yielded good sampleseparation and resolution from 123 to at least 1434 base pairs.

The invention has been described in detail with particular reference topreferred embodiments thereof, but it will be understood that variationsand modifications can be effected within the spirit and scope of theinvention.

We claim:
 1. A preformed, acrylamide based, water-soluble copolymeruseful for making an electrophoresis medium by crosslinking by areaction with a selected crosslinking agent that does not involve afree-radical vinyl addition mechanism, said copolymer comprising a minorproportion of a comonomer that contains a site for said crosslinkingreaction with said selected crosslinking agent, said preformed copolymerfurther having the following properties:1) M_(w) small enough that theaddition of 1.25 to 1.5 times the stoichiometric amount of the selectedcrosslinking agent will not raise the viscosity of the mixture so muchthat the medium cannot be poured into a 0.15×14×14 cm mold within about4 to 10 minutes after addition of the crosslinking agent, and 2) M_(n)large enough to provide a crosslink density sufficient to form a gelafter about 10 minutes after addition of 1.25 to 1.5 times thestoichiometric amount of the selected crosslinking agent, said gelultimately having sufficient integrity within about 2 hours afterpouring to be removed from the mold and handled gently without tearingor falling apart.
 2. The copolymer of claim 1 wherein the number ofequivalents of crosslinking sites per gram of copolymer is in the rangeof 0.45(10⁻⁴) to 14(10⁻⁴).
 3. The copolymer of claim 2 wherein saidnumber of equivalents is at least 2/M_(n).
 4. The copolymer of claim 2wherein said number of equivalents is from about 2.25(10⁻⁴) to 10(10⁻⁴).5. The copolymer of claim 1 wherein said M_(w) is such that saidaddition of said crosslinking agent will not raise the viscosity so muchthat the medium cannot be poured into said mold within about 6 to 8minutes after said addition.
 6. The copolymer of claim 2 wherein saidnumber of equivalents is from about 4(10⁻⁴) to 7(10⁻⁴).
 7. The copolymerof claim 1 which is a water soluble vinyl addition copolymer derivedfrom a mixture of monomers comprising from 85-98 mole percent of a firstmonomer selected from acrylamide and the N-substituted acrylamideswherein the N-substituent is an alkyl group having from 1 to 5 carbonatoms; from 2 to 15 mole percent of a second, vinyl monomer having areactive group selected from the group consisting of a) active halogengroups; b) activated 2-substituted ethylsulfonyl or activatedvinylsulfonyl groups; c) epoxy groups; d) isocyanate groups; e)aziridine groups; f) aldehyde groups; g) 2-substituted ethylcarbonylgroups; and h) succinimidoxycarbonyl groups; and from 0 to 12 molepercent of one or more other polymerizable nonionic vinyl monomersselected from styrene monomers, acrylic monomers, methacrylamidemonomers and N-substituted acrylamide monomers wherein the substituentcontains at least 6 carbon atoms.
 8. The copolymer of claim 7 whichcomprises from 90 to 97 mole percent of said first monomer.
 9. Thecopolymer of claim 7 which comprises from 3 to 10 mole percent of saidsecond monomer.
 10. The copolymer of claim 1 wherein said copolymer hasa number average molecular weight, M_(n), of at least about 7,000 and aweight average molecular weight, M_(w), of less than about 100,000. 11.The copolymer of claim 10 wherein said M_(n) is from about 7,000 toabout 30,000.
 12. The copolymer of claim 10 wherein said M_(w) is fromabout 25,000 to about 100,000.
 13. A method of preparing anelectrophoresis gel in situ which comprises mixing an aqueous solutionof a copolymer of claim 1 with a selected crosslinking agent that willreact with crosslinkable sites on said copolymer by a reaction with aselected crosslinking agent that does not involve a free-radical vinyladdition mechanism.
 14. The method of claim 13 wherein the number ofequivalents of crosslinking sites per gram of copolymer is in the rangeof 0.45(10⁻⁴) to 14(10⁻⁴).
 15. The method of claim 13 wherein saidcopolymer has a number average molecular weight, M_(n), of at leastabout 7,000 and a weight average molecular weight, M_(w), of less thanabout 100,000.
 16. The method of claim 13 wherein said M_(n) is fromabout 7,000 to about 30,000.
 17. The method of claim 13 wherein saidM_(w) is from about 25,000 to about 100,000.
 18. The method of claim 13wherein said copolymer is present in said solution in a concentration offrom about 8 to about 14% w/v.
 19. The method of claim 13 wherein saidcopolymer is present in said solution in a concentration of from about1.5 to about 6% w/v.
 20. The method of claim 18 wherein saidconcentration is from about 10 to 12%.
 21. The method of claim 19wherein said concentration is from about 2 to 5%.
 22. The method ofclaim 19 wherein said concentration is from about 2.5 to 4%.
 23. Themethod of claim 13 wherein the crosslinking agent is selected from thegroup consisting of compounds having two or more reactive groupsselected from amino, mercapto and phenolic hydroxy.
 24. The method ofclaim 13 wherein the crosslinking agent is selected from the groupconsisting of ethylenediamine, 1,3-propanediamine, 1,3-propanedithiol,dithiothreitol, dithioerythritol, 1,5-pentanediamine,hexamethylenediamine, diethylenetriamine, triethylenetetramine,propylenediamine, di(aminomethyl)ether, 1,8-diamino-4-(aminomethyl)octane, xylylenediamine, hydroquinone, bisphenol A, bisphenol sulfone,1,4-butanedisulfinic acid, benzenedisulfinic acid, thioethanolamine,p-aminothiophenol and butylenediamine.
 25. The method of claim 13wherein the crosslinking agent is dithiothreitol.
 26. An electrophoresiselement which comprises a resolving gel comprising a crosslinkedcopolymer of claim
 10. 27. An electrophoresis element which comprises astacking gel and a resolving gel, said stacking gel comprising acrosslinked copolymer of claim 13 and said resolving gel comprising acrosslinked copolymer of claim
 10. 28. A kit for electrophoresis whichcomprises a copolymer of claim 1, and, in a container separate from saidcopolymer, a crosslinking agent for crosslinking said copolymer by areaction that does not involve a free-radical vinyl addition mechanism.29. The kit of claim 28 which comprises a copolymer of claim
 10. 30. Thekit of claim 29 which further comprises a copolymer of claim
 13. 31. Thekit of claim 28 wherein said crosslinking agent is dithiothreitol.